Deoxynucleoside therapy for diseases caused by unbalanced nucleotide pools including mitochondrial dna depletion syndromes

ABSTRACT

The invention relates generally to a pharmacological therapy for human genetic diseases, specifically those characterized by unbalance nucleotide pools, more specifically mitochondrial DNA depletion syndromes, and more specifically, thymidine kinase 2 (TK2) deficiency. The pharmacological therapy involves the administration of at least one deoxynucleoside, or mixtures thereof. For the treatment of TK2 deficiency, the pharmacological therapy involves the administration of either deoxythymidine (dT) or deoxycytidine (dC), or mixtures thereof. This administration of deoxynucleosides is applicable to other disorders of unbalanced nucleotide pools, especially those found in mitochondrial DNA depletion syndrome.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. provisional patentapplication Ser. No. 62/180,194 filed Jun. 17, 2015, which is herebyincorporated by reference.

GOVERNMENT SUPPORT

This invention was made with government support under HD080642 awardedby NIH. The government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates generally to a pharmacological therapy for a humangenetic disease, specifically diseases characterized by unbalancednucleotide pools, e.g., mitochondrial DNA depletion syndromes, and morespecifically, thymidine kinase 2 (TK2) deficiency. The pharmacologicaltherapy involves the administration of at least one deoxynucleoside, ormixtures thereof. For the treatment of TK2 deficiency, thepharmacological therapy involves the administration of eitherdeoxythymidine (dT) or deoxycytidine (dC), or mixtures thereof. Thisadministration of one or more deoxynucleosides is applicable to otherdisorders of unbalanced nucleoside pools, especially those found inmitochondrial DNA depletion syndrome.

BACKGROUND OF THE INVENTION

Mitochondrial diseases are clinically heterogeneous diseases due todefects of the mitochondrial respiratory chain (RC) and oxidativephosphorylation, the biochemical pathways that convert energy inelectrons into adenosine triphosphate (ATP). The respiratory chain iscomprised of four multi-subunit enzymes (complexes I-IV) that transferelectrons to generate a proton gradient across the inner membrane ofmitochondria and the flow of protons through complex V drives ATPsynthesis (DiMauro and Schon 2003; DiMauro and Hirano 2005). CoenzymeQ₁₀ (CoQ₁₀) is an essential molecule that shuttles electrons fromcomplexes I and II to complex III. The respiratory chain is unique ineukaryotic, e.g., mammalian, cells by virtue of being controlled by twogenomes, mitochondrial DNA (mtDNA) and nuclear DNA (nDNA). As aconsequence, mutations in either genome can cause mitochondrialdiseases. Most mitochondrial diseases affect multiple body organs andare typically fatal in childhood or early adult life. There are noproven effective treatments for mitochondrial diseases, only supportivetherapies, such as the administration of CoQ₁₀ and its analogs toenhance respiratory chain activity and to detoxify reactive oxygenspecies (ROS) that are toxic by-products of dysfunctional respiratorychain enzymes.

Mitochondrial DNA depletion syndrome (MDS), which is a subgroup ofmitochondrial disease, is a frequent cause of severe childhoodencephalomyopathy characterized molecularly by reduction ofmitochondrial DNA (mtDNA) copy number in tissues and insufficientsynthesis of mitochondrial RC complexes (Hirano, et al. 2001). Mutationsin several nuclear genes have been identified as causes of infantileMDS, including: TK2, DGUOK, POLG, POLG2, SCLA25A4, MPV17, RRM2B, SUCLA2,SUCLG1, TYMP, OPA1, and C10orf2 (PEO1). (Bourdon, et al. 2007; Copeland2008; Elpeleg, et al. 2005; Mandel, et al. 2001; Naviaux and Nguyen2004; Ostergaard, et al. 2007; Saada, et al. 2003; Sarzi, et al. 2007;Spinazzola, et al, 2006). In addition, mutations in these nuclear genescan also cause multiple deletions of mtDNA with or without mtDNAdepletion (Bain, et al. 2012; Garone, et al. 2012; Langley, et al. 2006;Nishino, et al. 1999; Paradas, et al. 2012; Ronchi, et al. 2012;Spelbrink, et al. 2001; Tyynismaa, et al. 2009; Tyynismaa, et al. 2012;Van Goethem, et al. 2001).

One of these genes is TK2, which encodes thymidine kinase (TK2), amitochondrial enzyme required for the phosphorylation of the pyrimidinenucleosides (thymidine and deoxycytidine) to generate deoxythymidinemonophosphate (dTMP) and deoxycytidine monophosphate (dCMP) (Saada, etal. 2001). Mutations in TK2 impair the mitochondrialnucleoside/nucleotide salvage pathways required for synthesis ofdeoxynucleotide triphosphate (dNTP), the building blocks for mDNAreplication and repair.

TK2 deficiency was first described in 2001 by Saada and colleagues(Saada, et al. 2001), in four affected children originating from fourdifferent families, who suffered from severe, devastating myopathy.After an uneventful early development, at ages 6-36 months the patientsdeveloped hyperCKemia, severe muscle hypotonia with subsequent loss ofspontaneous activity. The disease was rapidly progressive and twopatients were mechanically ventilated at 3 years, while two otherpatients were already dead by the time of the report.

After the first description, sixty additional patients have beenreported in literature and at least twenty-six further patients havebeen diagnosed but not reported (Alston, et al, 2013; Bartesaghi, et al,2010; Behin, et al, 2012; Blakely, et al. 2008; Carrozzo, et al. 2003;Chanprasert, et al. 2013; Collins, et al. 2009; Galbiati, et al. 2006;Gotz, et al. 2008; Leshinsky-Silver, et al. 2008; Lesko, at al. 2010;Mancuso, et al. 2002; Mancuso, et al. 2003; Marti, at al. 2010; Oskoui,et al. 2006; Paradas, et al. 2012; Roos, et al. 2014; Tulinius, et al.2005; Tyynismaa, at al. 2012; Vila, et at 2003; Wang, et al. 2005),resulting in ninety patients, 53 males and 37 females.

The twenty-six patients recently diagnosed were identified throughnext-generation DNA sequencing. This large number of newly identifiedcases suggests that TK2 deficiency is an under diagnosed disorder.

TK2 deficiency manifests a wide clinical and molecular genetic spectrumwith the majority of patients manifesting in early childhood with adevastating clinical course, while others have slowly progressiveweakness over decades.

Treatment for TK2 deficiency, like most MDS and mitochondrial disorders,has been limited to supportive therapies. While the administration ofdeoxythymidine monophosphate (dTMP) and deoxycytidine monophosphate(dCMP) improved the conditions of both TK2 knock-in mutant mice andhuman patients with TK2 deficiency (U.S. application Ser. No.15/082,207, which is incorporated herein in its entirety), there isstill a need for therapeutic intervention for TK2 deficiency.

Additionally, there is a need for treatment for other forms of MDS andother diseases characterized by unbalanced nucleotide pools. Forexample, several mendelian disorders with mtDNA depletion or multipledeletions, or both are characterized by unbalanced deoxynucleotidetriphosphate pools that lead to defects of mtDNA replication. One suchdisorder, DGUOK mutations impair the intramitochondrial enzymedeoxyguanosine kinase, which normally phosphorylates the deoxypurinenucleosides deoxguanosine and deoxycytidine to generate deoxguanosinemonophosphate (dGMP) and deoxycytidine monophosphate (dCMP). Othernuclear genes that disrupt mitochondrial dNTP pools include TYMP, RRM2B,SUCLA2, SUCLG1 and MPV17. Therapies that restore dNTP pool balance wouldbe useful to treat these disorders as well.

SUMMARY OF THE INVENTION

In certain embodiments, the present invention relates to a method oftreating a disease or disorder characterized by unbalanced nucleotidepools, comprising administering to a subject in need thereof atherapeutically effective amount of a composition comprising one or moredeoxynucleosides.

Diseases or disorders characterized by unbalanced nucleotide pools thatcan be treated by the method of the current invention include, but arenot limited to, those characterized by mutations in the following genes:TK2; DGUOK; TYMP; RRM2B; SUCLA2; SUCLG1; and MPV17.

In a preferred embodiment, the disorder is a mitochondrial DNA depletionsyndrome (MDS). In a more preferred embodiment, the MDS includesdisorders of a myopathic form characterized by mutations in TK2, anencephalomyopathic form characterized by mutations in SUCLA2, aneurogastrointestinal encephalopathic form characterized by mutations inTYMP, and a hepatopathic form characterized by mutations in DGUOK, POLG,and MPV17. In a most preferred embodiment, the disorder is a thymidinekinase 2 deficiency, characterized by mutation(s) in the TK2 gene.

All mitochondrial DNA depletion syndromes can be treated with the methodof the current invention which comprises administering deoxynucleosides.Examples of MDS that can be treated by the method of the currentinvention include but are not limited to, deficiencies in the: DGUOKgene, encoding deoxyguanosine kinase, dGK; RRM2B gene, encoding p53R2,the p53 inducible small subunit of ribonucleotide reductase, RNR; andTYMP gene, encoding thymidine phosphorylase, TP.

In a preferred embodiment, the deoxynucleoside is either deoxythymidine(dT) or deoxycytidine (dC) or mixtures thereof. Deoxyadenosine (dA) anddeoxyguanosine (dG), alone or together, can also be used in the methodof the invention. One deoxynucleoside (i.e., dT, dC, dA, or dG) andmixtures of two or more of any of the four deoxynucleosides can be usedin the method of the invention.

Preferred dosages of the deoxynucleoside(s) are between about 100 andabout 1,000 mg/kg/day, more preferably between about 300 and about 800mg/kg/day, and most preferably between about 250 and about 600mg/kg/day. If the composition comprises a single deoxynucleoside, thenthe dosages are of the single deoxynucleoside. If the compositioncomprises more than one deoxynucleoside, the dosages can be of eachdeoxynucleoside or of the total deoxynucleosides in the composition.

Administration of the deoxynucleoside(s) can be once daily, twice daily,three times daily, four times daily, five times daily, up to six timesdaily, preferably at regular intervals.

Preferred methods of administration are oral, intrathecal, intravenous,and enteral.

Administration of the deoxynucleoside(s) should begin as soon as thedisorder characterized by unbalanced nucleotide pools, e.g., MDS, issuspected and continue throughout the life of the patient. Test for thediagnosis of such disorders including TK2 deficiency are known in theart.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purpose of illustrating the invention, there are depicted indrawings certain embodiments of the invention. However, the invention isnot limited to the precise arrangements and instrumentalities of theembodiments depicted in the drawings.

FIG. 1 depicts a growth curve of wild type (Tk2^(+/+) and Tk2⁺¹⁻), andTk2^(−/−) mice treated with 260 mg/kg/day or 520 mg/kg/day ofdeoxycytidine (dC) and deoxythymidine (dT) from postnatal day 4. Eachsymbol represents the mean of weight at each time-point. N of each groupis indicated in figure.

FIG. 2 depicts the survival curve of wild type (Tk2^(+/+)), andTk2^(−/−) mice with the following treatments: Tk2^(−/−milk) vsTk2^(−/−200 mg/kg/day dCMP+dTMP), p=0.0013; Tk2^(−/−milk) vsTk2^(−/−260 mg/kg/day dC+dT)p=0.0006; Tk2^(−/−milk) vsTk2^(−/−520 mg/kg/day dC+dT), p<0.0001; Tk2^(−/−260 mg/kg/day dC=dT) vsTk2^(−/−520 mg/kg/day dCdT), p=0.0009, at postnatal day 4. N of eachgroup indicated in figure. p-values determined by Mantel-Cox tests.

FIG. 3 are graphs of the relative proportions of dNTPs in isolatedmitochondria from brain and liver tissue of wild type (Tk2^(+/+)), andTk2^(−/−), untreated or treated with 200 mg/kg/day dCMP and dAMP, or 260mg/kg/day or 520 mg/kg/day of dcoxycytidinc (dC) and deoxythymidine (dT)at ages postnatal day 13 (top panels) and postnatal day 29 (bottompanels).

FIG. 4 are graphs showing the ratio of mtDNA/nDNA in brain, liver,intestine, and muscle in wild type Tk2 mice (Tk2^(−/−)) (left hand bar)as compared to Tk2^(−/−) mice, untreated or treated with 260 mg/kg/dayor 520 mg/kg/day of deoxycytidine (dC) and deoxythymidine (dT), at agespostnatal days 13 and 29. Data are represented as mean±standarddeviation (SD) of the percent of mtDNA copies relative to Tk2⁺. p-valueswere assessed by Mann-Whitney tests. (*p<0.05, ***p<0.001,****p<0.0001).

FIG. 5 are graphs depicting the results of HPLC measuring dT and uracilin plasma of untreated wild type (Tk2^(+/+)) mice, wild type (Tk2^(+/+))mice treated with 260 mg/kg/day of deoxycytidine (dC) and deoxythymidine(dT), Tk2^(−/−) mice treated with 260 mg/kg/day of deoxycytidine (dC)and deoxythymidine (dT), and Tk2^(−/−) mice treated with 200 mg/kg/dayof dCMP and dTMP, 30 minutes after treatment. Data arc expressed asmean±SD.

FIG. 6 are graphs of levels of respiratory chain enzyme activities inTk2^(−/−) mice treated with 400 mg/kg/day of dCMP and dTMP and THU at 13days postnatal, 260 mg/kg/day of deoxycytidine (dC) and deoxythymidine(dT) at 13 and 29 days postnatal, or 520 mg/kg/day of deoxycytidine (dC)and deoxythymidine (dT) 29 days postnatal. Data are represented as thepercent of the RCE activities in Tk2^(−/−) mouse tissues normalized toprotein levels and relative to Tk2⁺ for each treatment. p-valuesdetermined by Mann-Whitney tests. *p<0.05.

FIG. 7A is an immunoblot of respiratory chain proteins in wild type micetreated with 260 mg/kg/day or 520 mg/kg/day of deoxycytidine (dC) anddeoxythymidine (dT), and Tk2^(−/−) mice treated with 260 mg/kg/day or520 mg/kg/day of deoxycytidine (dC) and deoxythymidine (dT) at 29 dayspostnatal. FIG. 7B are graphs showing the RCE levels normalized tocomplex II, represented as percent of the RCE levels in TK2^(+/+) mice.p-values were assessed by Mann-Whitney tests.

Abbreviations:CS=citrate synthase; CI=NADH-dehydrogenase; CII=succinatedehydrogenase; CIII=cytochrome c reductase; CIV=cytochrome c oxidase(COX); CI+III=NADH-cytochrome c reductase; CII+III=succinatedehydrogenase-cytochrome c reductase.

DETAILED DESCRIPTION OF THE INVENTION

The current invention is based upon the surprising discovery thatmitochondrial DNA depletion syndromes, including TK2 deficiency, can betreated with deoxynucleosides. As shown by the results herein, theadministration of deoxynucleosides greatly improved the condition inboth a mouse model of TK2 deficiency and human patients with TK2deficiency.

Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and thespecific context where each term is used. Certain terms are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the methods of the invention and howto use them. Moreover, it will be appreciated that the same thing can besaid in more than one way. Consequently, alternative language andsynonyms may be used for any one or more of the terms discussed herein,nor is any special significance to be placed upon whether or not a termis elaborated or discussed herein. Synonyms for certain terms areprovided. A recital of one or more synonyms does not exclude the use ofthe other synonyms. The use of examples anywhere in the specification,including examples of any terms discussed herein, is illustrative only,and in no way limits the scope and meaning of the invention or anyexemplified term. Likewise, the invention is not limited to itspreferred embodiments.

The term “subject” as used in this application means mammals. Mammalsinclude canines, felines, rodents, bovine, equines, porcines, ovines,and primates. Thus, the invention can be used in veterinary medicine,e.g., to treat companion animals, farm animals, laboratory animals inzoological parks, and animals in the wild. The invention is particularlydesirable for human medical applications

The term “patient” as used in this application means a human subject. Insome embodiments of the present invention, the “patient” is known orsuspected of having a disease or disorder characterized by unbalancednucleotide pools, mitochondrial disease, mitochondrial DNA depletionsyndrome, or TK2 deficiency.

The phrase “therapeutically effective amount” is used herein to mean anamount sufficient to cause an improvement in a clinically significantcondition in the subject, or delays or minimizes or mitigates one ormore symptoms associated with the disease or disorder, or results in adesired beneficial change of physiology in the subject.

The terms “treat”, “treatment”, and the like refer to a means to slowdown, relieve, ameliorate or alleviate at least one of the symptoms ofthe disease or disorder, or reverse the disease or disorder after itsonset.

The terms “prevent”, “prevention”, and the like refer to acting prior toovert disease or disorder onset, to prevent the disease or disorder fromdeveloping or minimize the extent of the disease or disorder, or slowits course of development.

The term “in need thereof” would be a subject known or suspected ofhaving or being at risk of having a disease or disorder characterized byunbalanced nucleotide pools, mitochondrial disease, mitochondrial DNAdepletion syndrome, or TK2 deficiency.

The term “agent” as used herein means a substance that produces or iscapable of producing an effect and would include, but is not limited to,chemicals, pharmaceuticals, biologics, small organic molecules,antibodies, nucleic acids, peptides, and proteins.

The term “deoxynucleoside” as used herein means deoxythymidine or dT,deoxycytidine or dC, deoxyadenosine or dA, and deoxyguanosine or dG. Thefull length name and common abbreviation for each will be usedinterchangeably. Such deoxynucleosides also include physiologicallyfunctional derivatives of the deoxynucleosides.

As used herein, the term “physiologically functional derivative” refersto a compound (e.g, a drug precursor) that is transformed in vivo toyield a deoxynucleoside. The transformation may occur by variousmechanisms (e.g., by metabolic or chemical processes), such as, forexample, through hydrolysis in blood. Prodrugs are such derivatives, anda discussion of the use of prodrugs is provided by T. Higuchi and W.Stella, “Pro-drugs as Novel Delivery Systems,” Vol. 14 of the A.C.S.Symposium Series, and in Bioreversible Carriers in Drug Design, ed.Edward B. Roche, American Pharmaceutical Association and Pergamon Press,1987.

As used herein “an adverse effect” is an unwanted reaction caused by theadministration of a drug. In most cases, the administration of thedeoxynucleosides caused no adverse effects. The most expected adverseeffect would be a minor gastrointestinal intolerance.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system, i.e., thedegree of precision required for a particular purpose, such as apharmaceutical formulation. For example, “about” can mean within 1 ormore than 1 standard deviations, per the practice in the art.Alternatively, “about” can mean a range of up to 20%, preferably up to10%, more preferably up to 5%, and more preferably still up to 1% of agiven value. Alternatively, particularly with respect to biologicalsystems or processes, the term can mean within an order of magnitude,preferably within 5-fold, and more preferably within 2-fold, of a value.Where particular values are described in the application and claims,unless otherwise stated, the term “about” meaning within an acceptableerror range for the particular value should be assumed.

Administration of Deoxynucleosides for the Treatment of MitochonodrialDNA Depletion Syndrome

Mitochondrial DNA (mtDNA) depletion syndrome (MDS) comprises severalsevere autosomal diseases characterized by a reduction in mtDNA copynumber in affected tissues.

Most of the MDS causative nuclear genes encode proteins that belong tothe mtDNA replication machinery or are involved in deoxyribonucleosidetriphosphate (dNTP) metabolism.

One form of MDS is thymidine kinase deficiency or TK2. TK2 encoded bythe nuclear gene, TK2, is a mitochondrial matrix protein thatphosphorylates thymidine and deoxycytidine nucleosides to generatedeoxythymidine monophosphate (dTMP) and deoxycytidine monophosphate(dCMP), which in turn, are converted to deoxynucleotide triphosphates(dNTPs) required for mitochondrial DNA synthesis. As discussed in thebackground section, autosomal recessive TK2 mutations cause devastatingneuromuscular weakness with severe depletion of mitochondrial DNA(mtDNA) in infants and children, as well as progressive externalophthalmoplegia with mtDNA multiple deletions in adults. Many patientscannot walk and require some type of mechanical ventilation and feedingtube. The central nervous system is variably involved in thesedisorders, with symptoms that include seizures, encephalopathy,cognitive impairment, and hearing loss. Less than 7% of patients livemore than 42 years.

Based on clinical and molecular genetics findings of patients thusdiagnosed, three disease presentations were identified: i)infantile-onset (<1 year-old) myopathy with onset of weakness in thefirst year of life with severe mtDNA depletion and early mortality; ii)childhood-onset (>1-11 years-old) myopathy with severe mtDNA depletion;and iii) late-onset myopathy (>12 years-old) with mild weakness at onsetand slow progression to loss of ambulation, respiratory insufficiency,or both, often with chronic progressive external ophthalmoparesis inadolescence or adulthood in association with mtDNA multiple deletions,reduced mtDNA copy number, or both. See generally Garone, et al., (2016)in preparation.

Attempts to study the pathogenesis and test therapies for TK2 deficiencyusing cultured fibroblasts from patients have been unsuccessful, becausethe replicating cells failed to manifest mtDNA depletion. In contrast, ahomozygous Tk2 H126N knock-in mutant (Tk2^(−/−)) mouse model, manifestsa phenotype that is strikingly similar to the human infantileencephalomyopathy caused by TK2 mutations, characterized by onset at age10 days with decreased ambulation, unstable gait, coarse tremor, growthretardation, and depletion of mitochondrial DNA (mtDNA) progressingrapidly to early death at age 14 to 16 days, which is a time periodanalogous to the human infantile-onset disease (Akman, et al. 2008;Dorado, et al. 2011).

The studies set forth herein with Tk2 knock-in mice have shown theadministration of oral dC/dT prolonged delayed the onset of clinicalsymptoms of TK2 deficiency and prolonged the lives of the mice by two-to three-fold (Example 2).

Additional experiments showed tissue-specific effects. Measurement ofthe dNTP pool levels in mitochondria extracts showed that dCTP wasrescued in brain and dTTP was rescued in liver (Example 3). Measurementof mtDNA depletion showed both dCMP+dTMP and dC+dT therapies rescued themtDNA copy number in liver, muscle and tissue (Example 4). It waspreviously speculated that formation of the blood brain barrier might becompromising the treatment bioavailability in brain. Nevertheless, HPLCmeasurements showed that catalytic products of these compounds werefound in higher concentrations after both nucleotides monophosphates anddeoxynucleosides treatment, suggesting that they are capable of crossingthe blood brain barrier. mtDNA depletion measurements also showed acompletely rescue of mtDNA copy number in intestine.

Thus, the experiments set forth herein using the mouse model of Tk2deficiency show the administration of deoxynucleosides to be effectiveand safe for the treatment of the disease. Additionally, as shown inExample 5, the administration of dT and dC greatly improved the symptomsof TK2 deficiency in patients.

Thus, the present invention includes the administration of at least onedeoxynucleoside to a patient in need thereof. In one embodiment, thepresent invention includes the administration of at least onedeoxpyrimidine. In a further embodiment, the deoxypyrimidine is chosenfrom dC, dT and mixtures thereof. In yet another embodiment, the presentinvention includes the administration of at least one deoxypurine. In afurther embodiment, the deoxypurine is chosen from dA, dG, and mixturesthereof.

Patients who would benefit from the administration of deoxynucleosideswould be those diagnosed with TK2 deficiency. In these patients, atleast one deoxypyrimidine, dC or dT, or mixtures thereof would beadministered.

A parallel defect of deoxyguanosine kinase (dGK), due to autosomalrecessive mutations in DGUOK with deficiencies in dGMP and dAMP, causesmtDNA depletion typically manifesting as early childhood-onsethepatocerebral disease (Mandel, et al. 2001). These patients wouldbenefit from the administration of at least one deoxypurine, dG or dA,or mixtures thereof.

Other forms of MDS as well as other disorders related to unbalancednucleotide pools can be treated by the administration of specificdeoxynucleosides, i.e., dA, dG, dC, or dT, or mixtures thereof. Thesedisorders would include but are not limited to deficiencies related toRRM2B (encoding p53R2, the p53 inducible small subunit of ribonucleotidereductase, RNR) and mutations in TYMP (encoding thymidine phosphorylase,TP) which cause mitochondrial neurogastrointestinal encephalomyopathy(MNGIE). Additional nuclear genes that disrupt mitochondrial dNTP poolsinclude but are not limited to SUCLA2, SUCLG1 and MPVI7. Disordersrelated to these genes can also be treated by the administration of oneor more deoxynucleosides.

Additionally, as the mechanisms of other forms of MDS and otherdisorders become elucidated, the proper deoxynucleoside(s) for treatmentcan be determined by the skilled practitioner.

Patients that exhibit the phenotype discussed above for TK2 deficiencyincluding the most typical presentation of progressive muscle diseasecharacterized by generalized hypotonia, proximal muscle weakness, lossof previously acquired motor skills, poor feeding, and respiratorydifficulties, can be tested to definitively diagnose the disease.

If the clinical presentation is highly suspicious for mtDNA depletionsyndrome, molecular genetic testing using a panel of genes known tocause mtDNA depletion syndrome should be performed (Chanprasert, et al.2012). The TK2 gene is the only gene in which mutations are known tocause TK2-related mitochondrial DNA depletion syndrome. This testing caninclude a sequence analysis of the entire coding and exon/intronjunction regions of TK2 for sequence variants and deletion/duplication.If compound heterozygous or homozygous deleterious mutations areidentified in the sequence analysis, the diagnosis of TK2 deficiency isconfirmed, and thus, the subject would benefit from the deoxynucleosidetherapy. If sequence analysis does not identify two compoundheterozygous or homozygous deleterious mutations, deletion/duplicationanalysis should be considered to determine and/or confirm a TK2deficiency diagnosis.

Further tests to determine and/or confirm a TK2 deficiency diagnosis mayinclude testing serum creatine kinase (CK) concentration,electromyography, histopathology on skeletal muscle, mitochondrial DNA(mtDNA) content (copy number), and electron transport chain (ETC)activity in skeletal muscle. If one or more of the following is found inthese tests, the TK2 deficiency is determined and/or confirmed. ElevatedCK concentration as compared to healthy controls can indicate TK2deficiency. A skeletal muscle biopsy can be performed, and then a mtDNAcontent analysis in skeletal muscle performed. If the skeletal musclebiopsy shows prominent variance in fiber size, variable sarcoplasmicvacuoles, variable increased connective tissue, and ragged red fibers aswell as increased succinate dehydrogenase (SDH) activity and low toabsent cytochrome c oxidase (COX) activity, and mtDNA copy number isseverely reduced (typically less than 20% of age- and tissue-matchedhealthy controls), a diagnosis of TK2 deficiency can be determinedand/or confirmed (Chanprasert, et al. 2012).

Additionally, TK2 deficiency is inherited in an autosomal recessivemanner. Thus, a sibling of an affected patient can be tested as early aspossible after birth to diagnose the disease.

In all of these examples, deoxynucleoside therapy should be started assoon as possible after a diagnosis of TK2 deficiency.

Pharmaceutical Compositions, Methods of Administration, and Dosing

The present invention encompasses the administration ofdeoxynucleosides, more specifically one or more deoxynucleosides.

Most preferred methods of administration are oral, intrathecal andparental including intravenous. The deoxynucleosides must be in theappropriate form for administration of choice.

Deoxynucleosides are easily dissolved in liquid are easily dissolved inliquid (such as water, formula or milk) whereas the free acid form doesnot readily dissolve in liquid.

Such pharmaceutical compositions comprising one of more deoxynucleosidesfor administration may comprise a therapeutically effective amount ofthe deoxynucleosides and a pharmaceutically acceptable carrier. Thephrase “pharmaceutically acceptable” refers to molecular entities andcompositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human, and approved by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopeia for use inanimals, and more particularly in humans. “Carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas saline solutions in water and oils, including those of petroleum,animal, vegetable, or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil, and the like. A saline solution is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol, and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents.

Oral administration is a preferred method of administration. Thedeoxynucleosides can be added to any form of liquid a patient wouldconsume including but not limited to, milk, both cow's and human breast,infant formula, and water.

Additionally, pharmaceutical compositions adapted for oraladministration may be capsules, tablets, powders, granules, solutions,syrups, suspensions (in non-aqueous or aqueous liquids), or emulsions.Tablets or hard gelatin capsules may comprise lactose, starch orderivatives thereof, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, stearic acid or salts thereof. Soft gelatincapsules may comprise vegetable oils, waxes, fats, semi-solid, or liquidpolyols. Solutions and syrups may comprise water, polyols, and sugars.An active agent intended for oral administration may be coated with oradmixed with a material that delays disintegration and/or absorption ofthe active agent in the gastrointestinal tract. Thus, the sustainedrelease may be achieved over many hours and if necessary, the activeagent can be protected from degradation within the stomach.Pharmaceutical compositions for oral administration may be formulated tofacilitate release of an active agent at a particular gastrointestinallocation due to specific pH or enzymatic conditions.

In order to overcome any issue of the deoxynucleosides crossing theblood/brain barrier, intrathecal administration is a further preferredform of administration (Galbiati, et al. 2006; Gotz, et al. 2008).Intrathecal administration involves injection of the drug into thespinal canal, more specifically the subarachnoid space such that itreaches the cerebrospinal fluid. This method is commonly used for spinalanesthesia, chemotherapy, and pain medication. Intrathecaladministration can be performed by lumbar puncture (bolus injection) orby a port-catheter system (bolus or infusion). The catheter is mostcommonly inserted between the laminae of the lumbar vertebrae and thetip is threaded up the thecal space to the desired level (generallyL3-L4). Intrathecal formulations most commonly use water, and saline asexcipients but EDTA and lipids have been used as well.

A further preferred form of administration is parenteral includingintravenous administration. Pharmaceutical compositions adapted forparenteral administration, including intravenous administration, includeaqueous and non-aqueous sterile injectable solutions or suspensions,which may contain anti-oxidants, buffers, bacteriostats, and solutesthat render the compositions substantially isotonic with the blood ofthe subject. Other components which may be present in such compositionsinclude water, alcohols, polyols, glycerine, and vegetable oils.Compositions adapted for parental administration may be presented inunit-dose or multi-dose containers, such as sealed ampules and vials,and may be stored in a freeze-dried (lyophilized) condition requiringonly the addition of a sterile carrier, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules, and tablets. Suitable vehicles that can beused to provide parenteral dosage forms of the invention are well knownto those skilled in the art. Examples include: Water for Injection USP;aqueous vehicles such as Sodium Chloride Injection, Ringer's Injection,Dextrose Injection, Dextrose and Sodium Chloride Injection, and LactatedRinger's Injection; water-miscible vehicles such as ethyl alcohol,polyethylene glycol, and polypropylene glycol; and non-aqueous vehiclessuch as corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate,isopropyl myristate, and benzyl benzoate.

Additionally, since some patients may be receiving enteral nutrition bythe time the deoxynucleoside treatment begins, the dNs can beadministered through a gastronomy feeding tube or other enteralnutrition means.

Further methods of administration include mucosal, such as nasal,sublingual, vaginal, buccal, or rectal; or transdermal administration toa subject.

Pharmaceutical compositions adapted for nasal and pulmonaryadministration may comprise solid carriers such as powders, which can beadministered by rapid inhalation through the nose. Compositions fornasal administration may comprise liquid carriers, such as sprays ordrops. Alternatively, inhalation directly through into the lungs may beaccomplished by inhalation deeply or installation through a mouthpiece.These compositions may comprise aqueous or oil solutions of the activeingredient. Compositions for inhalation may be supplied in speciallyadapted devices including, but not limited to, pressurized aerosols,nebulizers or insufflators, which can be constructed so as to providepredetermined dosages of the active ingredient.

Pharmaceutical compositions adapted for rectal administration may beprovided as suppositories or enemas. Pharmaceutical compositions adaptedfor vaginal administration may be provided as pessaries, tampons,creams, gels, pastes, foams or spray formulations.

Pharmaceutical compositions adapted for transdermal administration maybe provided as discrete patches intended to remain in intimate contactwith the epidermis of the recipient over a prolonged period of time.

The deoxynucleoside therapy comprises the administration of one or moredeoxynucleosides chosen from the group consisting of deoxythymidine(dT), deoxycytidine (dC), deoxyadenosine (dA) and deoxyguanosine (dG).

A skilled practitioner can determine which deoxynucleosides arebeneficial based upon the deficiency. It is also within the skill of theart for the practitioner to determine if mixtures of thedeoxynucleosides should be administered and in what ratio. If twodeoxynucleosides are to be administered, they can be in a ratio of 50/50of each deoxynucleoside, e.g., dC and dT, or in ratios of about 5/95,10/90, 15/85, 20/80, 25/75, 30/70, 35/65, 40/60, 45/55, 55/45, 60/40,65/35, 70/30, 75/25, 80/20, 85/15, 90/10, and 95/5.

By way of example, dT and dC arc administered in mixture of equalamounts for TK2 deficiency.

Selection of a therapeutically effective dose will be determined by theskilled artisan considering several factors, which will be known to oneof ordinary skill in the art. Such factors include the particular formof the deoxynucleoside, and its pharmacokinetic parameters such asbioavailability, metabolism, and half-life, which will have beenestablished during the usual development procedures typically employedin obtaining regulatory approval for a pharmaceutical compound. Furtherfactors in considering the dose include the condition or disease to betreated or the benefit to be achieved in a normal individual, the bodymass of the patient, the route of administration, whether theadministration is acute or chronic, concomitant medications, and otherfactors well known to affect the efficacy of administered pharmaceuticalagents. Thus, the precise dose should be decided according to thejudgment of the person of skill in the art, and each patient'scircumstances, and according to standard clinical techniques.

A preferred dose ranges from about 100 mg/kg/day to about 1,000mg/kg/day. A further preferred dose ranges from about 200 mg/kg/day toabout 800 mg/kg/day. A further preferred dose ranges from about 250mg/kg/day to about 400 mg/kg/day. These dosage amounts are of individualdeoxynucleosides or of a composition with a mixture of more than onedeoxynucleosides, e.g., dT and dC. For example, a dose can comprise 400mg/kg/day of dT alone. In a further example, a dose can comprise amixture of 200 mg/kg/day of dT and 200 mg/kg/day of dC. In a furtherexample, a dose can comprise 400 mg/kg/day of a mixture of dT and dC.

Administration of the deoxynucleosides can be once a day, twice a day,three times a day, four times a day, five times a day, up to six times aday, preferably at regular intervals. For example, when thedeoxynucleosides are administered four times daily, doses would be at8:00 AM, 12:00 PM, 4:00 PM, and 8:00 PM.

Doses can also be lowered if being administered intravenously orintrathecally. Preferred dose ranges for such administration are fromabout 50 mg/kg/day to about 500 mg/kg/day.

As shown in Example 5, doses can be adjusted to optimize the effects inthe subject. For example, the deoxynucleosides can be administered at100 mg/kg/day to start, and then increased over time to 200 mg/kg/day,to 400 mg/kg/day, to 800 mg/kg/day, up to 1000 mg/kg/day, depending uponthe subject's response and tolerability.

A subject can be monitored for improvement of their condition prior toincreasing the dosage. A subject's response to the therapeuticadministration of the deoxynucleosides can be monitored by observing asubject's muscle strength and control, and mobility as well as changesin height and weight. If one or more of these parameters increase afterthe administration, the treatment can be continued. If one or more ofthese parameters stays the same or decreases, the dosage of thedeoxynucleosides can be increased.

As shown in the Examples, the deoxynucleosides are well tolerated. Anyobserved adverse effects were minor and were mostly diarrhea, abdominalbloating and other gastrointestinal manifestations. A subject can alsobe monitored for any adverse effects, such as gastrointestinalintolerance, e.g., diarrhea. If one or more adverse effects are observedafter administration, then the dosage can be decreased. If no suchadverse effects are observed, then the dosage can be increased.Additionally, once a dosage is decreased due to the observation of anadverse effect, and the adverse effect is no longer observed, the dosagecan be increased.

The deoxynucleosides can also be co-administered with other agents. Suchagents would include therapeutic agents for treating the symptoms of theparticular form of MDS. In particular, for TK2 deficiency, the dT and dCcan be co-administered with an inhibitor of ubiquitous nucleosidecatabolic enzymes, including but not limited to enzyme inhibitors suchas tetrahydrouridine (inhibitor of cytidine deaminase) and immucillin H(inhibitor of purine nucleoside phosphorylase) and tipiracil (inhibitorof thymidine phosphorylase). Such inhibitors are known and used in thetreatment of some cancers.

EXAMPLES

The present invention may be better understood by reference to thefollowing non-limiting examples, which are presented in order to morefully illustrate the preferred embodiments of the invention. They shouldin no way be construed to limit the broad scope of the invention.

Example 1—Materials and Methods

Mouse Model of TK2 Deficiency

A homozygous Tk2 H126N knock-in mutant (Tk2^(−/−)) mouse that manifestsa phenotype strikingly similar to the human infantile encephalomyopathyhas been previously reported (Akman, et al. 2008). Between postnatal day10 and 13, Tk2^(−/−) mice rapidly develop fatal encephalomyopathycharacterized by decreased ambulation, unstable gait, coarse tremor,growth retardation, and rapid progression to early death at age 14 to 16days. Molecular and biochemical analyses of the mouse model demonstratedthat the pathogenesis of the disease is due to loss of enzyme activityand ensuing dNTP pool imbalances with decreased dTTP levels in brain andboth dTTP and dCTP levels in liver, which, in turn, produces mtDNAdepletion and defects of respiratory chain enzymes containingmtDNA-encoded subunits, most prominently in the brain and spinal cord.

All experiments were performed according to a protocol approved by theInstitutional Animal Care and Use Committee of the Columbia UniversityMedical Center, and were consistent with the National Institutes ofHealth Guide for the Care and Use of Laboratory Animals. Mice werehoused and bred according to international standard conditions, with a12-hour light, 12-hour dark cycle, and sacrificed at 4, 13, and 29 daysof age.

Organs (brain, spinal cord, liver, heart, kidney, quadriceps muscle,lung, and gastrointestinal tract) were removed and either frozen in theliquid phase of isopentane, pre-cooled near its freezing point (−160°C.) with dry ice or fixed in 10% neutral buffered formalin and embeddedin paraffin using standard procedures. Paraffin embedded tissue werethen stained with hematoxylin and eosin (H&E) for morphological study orprocessed for immunostaining studies with GFAP, COX I, or complex Isubunit as detailed described in the supplemental procedures. Bothheterozygous and homozygous wild type mice were considered as controlgroup (Tk2′) since no clinical and biochemical difference werepreviously described (Akman, et al. 2008; Dorado, et al. 2011).

Treatment Administration and Experimental Plan

Deoxycytidine (dC) and deoxythymidine (dT) were administered in 50 μl ofEsbilac milk formula for small pets (Pet-Ag) by daily oral gavage to Tk2H126N knockin mice (Tk2^(−/−)) and aged matched control wild-type (Tk2⁺)using 2 doses, 260 mg/kg/day and 520 mg/kg/day, from post-natal day 4 to29 days. At age 21 days, mice were separated from the mother and thetreatment was continued by administration of dC and dT in drinking waterusing equimolar doses respectively of 1.6 mM and 3.2 mM. A negativecontrol group of untreated Tk2 mutant and control wild-type mice wereweighed and observed closely for comparison.

Phenotype Assessment

Body weight was assessed daily, since it has been previously observedthat incapacity of gaining weight is the first sign of disease (Akman,et al. 2008).

To define the degree of safety and efficacy of dT/dC, survival time,age-at-onset of disease, type and severity of symptoms, occurrence ofside effects, and proportion of treatment termination due to adverseevents in treated and untreated Tk2 mice were compared. Generalbehavior, survival time, and body weights of the mice were assesseddaily beginning at postnatal day 4.

dNTP Pool by Polymerase Extension Assay

Tissues were homogenized on ice in 10 volumes (w/v) of cold MTSE buffer(210 mM mannitol, 70 mM sucrose, 10 mM Tris-HCl pH 7.5, 0.2 mM EGTA,0.5% BSA) and centrifuged at 1000 g for 5 minutes at 4° C., followed bythree centrifugations at 13,000 g for 2 minutes at 4° C. Supernatant wasprecipitated with 60% methanol, kept 2 hours at −80° C., boiled 3minutes, stored at −80° C. (from 1 hour to overnight) and centrifuged at20,800 g for 10 minutes at 4° C. Supernatants were evaporated until dryand pellet was resuspended in 65 R1 of water and stored at −80° C. untilanalysed. To minimize ribonucleotide interference, total dNTP pools weredetermined as reported (Ferraro, et al. 2010; Marti, et al. 2012a).Briefly, 20 μl volume reactions was generated by mixing 5 μl of sampleor standard dNTP with 15 μl of reaction buffer [0.025 U/mlThermoSequenase DNA polymerase (GE Healthcare, Piscataway, N.J., USA) orTaq polymerase (Life Technologies, N.Y., USA), 0.75 μM 3H-dTTP or3H-dATP (Moravek Biochemicals), 0.25 μM specific oligonucleotide, 40 mMTris-HCl, pH 7.5, 10 mM MgCl2, 5 mM DTT]. After 60 minutes at 48° C., 18ml of reaction were spotted on Whatman DE81 filters, air dried andwashed three times for 10 minutes with 5% Na2HPO4, once in distilledwater and once in absolute ethanol. The retained radioactivity wasdetermined by scintillation counting.

Nucleosides Measurements by HPLC

Deoxythymidine (dT), deoxyuridine (dU), uracil (U) and thymine (T)levels were assessed by a gradient-elution HPLC method as describedpreviously (Lopez, et al. 2009; Marti, et al. 2012b), with minormodifications. Briefly, deproteinized samples were injected into anAlliance HPLC system (Waters Corporation) with an Alltima C18NUCreversed-phase column (Alltech) at a constant flow rate of 1.5 ml/min(except where indicated) using four buffers: eluent A (20 mM potassiumphosphate, pH 5.6), eluent B (water) and eluent C (methanol). Sampleswere eluted over 60 minutes with a gradient as follows: 0-5 min, 100%eluent A; 5-25 min. 100-71% eluent A, 29% eluent B; 25-26 min, 0-100%eluent C; 26-30 min, 100% eluent C; 30-31 min, 0-100% eluent B; 31-35min, 100% eluent B (1.5-2 ml/min); 35-45 min, 100% eluent B (2 ml/min);45-46 min, 100% eluent B (2-1.5 ml/min); 46-47 min, 0-100% eluent C;47-50 min. 100% clucnt C; 50-51 min, 0-100% clucnt A; and 51-60 min,100% clucnt A.

Absorbance of the eluates was monitored at 267 nm and dThd and dUrdpeaks were quantified by comparing their peak areas with a calibrationcurve obtained with aqueous standards. For definitive identification ofdeoxythymidine, deoxyuridine, uracil, and thymine peaks for each sample,a second aliquot was treated with excess of purified E. coli TP (Sigma)to specifically eliminate dT and dU. The detection limit of this methodis 0.05 mmol/1 for all nucleosides. Results were expressed as nmol/mg ofprotein.

RT-qPCR: Mitochondrial DNA Quantification

Real-time PCR was performed with the primers and probes for murine COX Igene (mtDNA) and mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH,nDNA) (Applied Biosystems, Invitrogen, Foster City, Calif., USA) asdescribed as described using ddCt method in a Step One Plus Real TimePCR System (Applied Biosystems) (Dorado, et al. 2011). MtDNA values werenormalized to nDNA values and expressed as percent relative to wild-type(100%).

Mitochondrial Respiratory Chain Protein Levels

Thirty micrograms of whole brain cerebrum or cerebellum extracts wereelectrophoresed in an SDS-12% PAGE gel, transferred to Immun-Blot™ PVDFmembranes (Biorad, Hercules, Calif., USA) and probed with MitoProfile®Total OXPHOS Rodent WB Antibody Cocktail of antibodies (MitoSciences,Eugene, Oreg., USA). Protein-antibody interaction was detected withperoxidase-conjugated mouse anti-mouse IgG antibody (Sigma-Aldrich, StLouis, Mo., USA), using Amersham™ ECL Plus western blotting detectionsystem (GE Healthcare Life Sciences, UK). Quantification of proteins wascarried out using NIH ImageJ 1.37V software. Average gray value wascalculated within selected areas as the sum of the gray values of allthe pixels in the selection divided by the number of pixels.

Mitochondrial Respiratory Chain Enzyme Activities by SpectrophotometerAnalysis

Mitochondrial RC enzymes analysis was performed in cerebrum tissue aspreviously described (DiMauro, et al. 1987).

Statistical Methods

Data are expressed as the mean±SD of at least 3 experiments per group.Gehan-Breslow-Wilcoxon test was used to compare the survival proportionof each group of mice. A p-value of <0.05 was considered to bestatistically significant.

Example 2—The Administration of dC/dT to Tk2^(−/−) Mice Delayed theClinical Onset of TK2 Deficiency and Increased Survival

A dose of 260 and 520 mg/kg/day each of deoxynucleosides (dC/dT) wereadministered to the Tk2^(−/−) mice. These doses of deoxynucleosides werethe molar equivalent of 400 and 800 mg/kg/day of dCMP+dTMP respectively.

Mice treated with oral dC+dT (260 or 520 mg/kg/day from age 4 days)appeared normal until postnatal day 21 (FIG. 1). After age 21 days,mutant mice treated with 260 mg/kg/day dose(Tk2^(−/−260 mg/kg/day dC/dT)) stopped gaining weight and developed mildhead tremor and weakness that led to death at postnatal day 31±4.3 (FIG.2).

Mutant mice treated with the 520 mg/kg/day dC+dT(Tk2^(−/−520 mg/Kg/day dC/dT)) continued to gain weight for oneadditional week, but subsequently manifested deterioration similar toTk2^(−/−260 mg/Kg/day dC/dT), and died at postnatal day 43±10. Theseresults are comparable to those showed by Tk2^(−/−) mice treated with200 or 400 mg/kg/day of oral dCMP/dTMP treatment.Tk2^(+260 mg/kg/day dc/dT) and Tk2^(+520 mg/kg/day dc/dT) were followeduntil postnatal day 60. No side effects were observed.

As shown, the lifespan of the treated Tk2^(−/−) was significantlyincreased. Untreated Tk2^(−/−) mice showed a mean lifespan of 13 days,while treated mice survived a mean of 31 and 40 days with the 260 and520 mg/kg/day dose, respectively (FIG. 2). Interestingly, one of themice survived to postnatal day 56, which has been the longest lifespanfor the Tk2 knock-in mouse model to date.

Example 3—Oral dC/dT Ameliorates Molecular Abnormalities in Brain andLiver

Measurement of dNTPs in mitochondrial extract showed that bothTk2^(−/−260 mg/Kg/day dC/dT) and Tk2^(−/−520 mg/Kg/day dC/dT) did notfully correct mitochondrial dNTP pool imbalances at postnatal day 13 andmanifested variable effects in tissues with a completed rescue of dCTPdeficits in brain, while dTTP was corrected in the liver. In contrast,deficiencies of dTTP in brain and dCTP in liver remained severe despitedeoxynucleoside supplementation (FIG. 3).

In Tk2^(−/−260 mg/Kg/day dC/dT) and Tk2^(−/−520 mg/Kg/day dC/dT) mice atpostnatal day 13, the treatment prevented mtDNA depletion in heart,liver, kidney, intestine and muscle (FIG. 4). In contrast, mtDNA copynumber was only partially ameliorated in brain at postnatal day 13 in adose-dependent manner with mtDNA/nDNA ratios relative to control brainreaching 39% with 260 mg/kg/day of dC+dT and 52% with 520 mg/kg/day.Measurements of the bases dT and uracil in brain by HPLC showed higherlevels in animals treated with dC+dT or with dCMP+dTMP (FIG. 5), furtherindicating that both deoxynucleosides and deoxynucleoside monophosphatescross the blood brain barrier. At postnatal day 29, mtDNA depletion waspartially rescued by 260 and 520 mg/kg/day of dC+dT therapy in heart (40and 35%), liver (46 and 45%), kidney (38 and 42%) and muscle (24 and35%), but strikingly was fully rescued in intestine (82 and 84%) (FIG.4).

Example 4—Oral dC/dT Ameliorates Biochemical Abnormalities in Brain

Respiratory chain enzyme (RCE) activities and protein levels werecompletely rescued in brain of TK2^(−/−260 mg/Kg/day dC/dT) at postnatalday 13 (FIG. 6). RCE activities were also restored at postnatal day 29,and only a slight decrease of complex I activity could be observed inTK2^(−/−520 mg/Kg/day dC/dT) (FIG. 6). RCE protein levels in brain werepartially restored at postnatal day 29 with higher levels inTK2^(−/−520 mg/Kg/day dC/dT) than in TK2^(−/−260 mg/Kg/day dC/dT) (FIG.7). These differences in protein levels were consistent with thedifferences in mtDNA depletion in brains of treated mutant mice atpostnatal day 29, and likely accounted for the prolonged survivalobserved with the higher dose.

Example 5—Administration of dC/dT in Patients with TK2 Deficiency WasEfficacious

Symptoms, dosages, and outcomes of patients with TK2 deficiency who havereceived deoxynucleoside therapy under the supervision and control ofthe inventors are summarized below.

Patient 1

This patient was horn in the United States in February 2011. Hissymptoms manifested at 12 months with hypotonic and a floppy head. Hehas never walked. He also has respiratory muscle weakness and was put onmechanical ventilation at 19 months, of which he is still on 24hours/day. He has also been on a feeding tube since 19 months.

He was previously on 100 mg/kg/day and then 200 mg/kg/day of dCMP anddTMP. On this therapy, he was able to grip small objects and his weightincreased from 10.4 kg to 19.5 kg.

In October of 2015, he began on 260 mg/kg/day of dC and dT which wasincreased to 340 mg/kg/day of dC and dT. After two months, he was movinghis hands and head better, able to stand 5 minutes with support of aperson, starting to cough, and his heart rate was slower (down from140-170 bpm during day, to 100-120 bpm during day).

On Mar. 23, 2016, the dose was increased to 400 mg/kg/day of dC and dT.After 6 weeks on this therapy, he showed further improvements: he wasable to sit in a chair about 5 hours/day; stood in a “Stander” for 1.5hours; about to grab and hold small stuffed animals; pressed computerbuttons; untied his diapers and aimed his penis to wet the personchanging the diaper; and held his knees flexed for a few seconds.

The only adverse effect seen during the treatment was diarrhea.

Patient 2

This patient was born in Spain in 1987. He began showing symptoms at 3years of age including proximal muscle weakness. He lost the ability towalk at age 13 and was ventilated 24 hours a day. He was previouslytaking dAMP and dCMP at 200 mg/kg/day and showed a weight increase and adecrease of 24 to 22 hours a day on ventilation.

He has been on deoxynucleoside therapy since June of 2015 at 400mg/kg/day dC and dT, and has shown improvement in muscle strength, hisweight and ventilation have stabilized, and he is enjoying a betterquality of life.

The only adverse effects seen during the treatment was diarrhea and hairloss.

Patient 3

This patient was born in Spain in 1985. His symptoms began at 6 yearsold with facial, proximal, and axial muscle weakness. He started 200mg/kg/day of dT and dC in June of 2015 and to date, his condition hasimproved with improvements in 6 minute walk test, time to get up and go,and climb up and down 4 steps.

The only adverse effect seen during the treatment was diarrhea.

Patient 4

This patient was born in Spain in February 2009. His symptoms manifestedat six months with failure to thrive. He started on 230 mg/kg/day of dCand dT in July of 2015. By January of 2016, he showed improvement in hiscondition and was eating better.

There were no observed adverse effects.

Patient 5

This patient was born in Spain in 1957 and began to have symptoms at 50years old of orthopnea, and diaphragmatic weakness. He is on BiPAP atnight. He started on 200 mg/kg/day of dC and dT in November of 2015.

There were no observed adverse effects.

Patient 6

This patient was born in Spain in October 2011, and starting showingsymptoms at 15 months, including hypotonia and weakness. He lostambulation at 22 months, and has respiratory muscle weakness. He startedmechanical ventilation at 16 months and is currently on BiPAP twelvehours a day. He was previously on dCMP and dAMP at 100 mg/kg/day thatwas increased to 400 mg/kg/day. His strength as shown by EgenKlassification scale improved (28/30 to 13/30) and his weight increasedfrom 9.8 kg to 12.3 kg.

He began deoxynucleoside therapy in April 2015 at 400 mg/kg/day of dCand dT. In October of 2015, his change in Egen Klassification scale wentfrom 13/30 to 11/30 and his weight increased to 16.5 kg from 12.3 kg.

There were no observed adverse effects.

Patient 7

This patient was born in Spain in November of 2012. He started showingsymptoms at 17 months including weakness and hypotonia. He lostambulation at 22 months and started mechanical ventilation at 29 months.He was previously on dCMP and dAMP at 100 mg/kg/day that was increasedto 400 mg/kg/day. His strength as shown by Egen Klassification scaleimproved (30/30 to 24/30) and his weight increased from 11 kg to 15.7kg.

He started deoxynucleoside therapy in April of 2015 with a dose of 400mg/kg/day dT and dC. In November of 2015, his change in EgenKlassification scale went from 24/30 to 19/30 and his weight increasedto 17 kg from 15.7 kg.

There were no observed adverse effects.

Patient 8

This patient was born in Chile in September of 1989 and started showingsymptoms at 11 months with frequent falls and progressive gaitimpairment. She lost the ability to walk alone at about 4 years of age.She had been on nucleotide therapy previously and showed improvement inher mobility, including walking unassisted, standing longer, climbingstairs, attending gym class, and attending to personal needs.

She switched to deoxynucleoside therapy in February of 2016 at a dose of260 mg/kg/day of dC and dT, and then increased to a dose of 400mg/kg/day of dC and dT in May of 2016 and continued to show improvement.

There were no observed adverse effects.

Patient 9

This patient was born in Guatemala in September of 1989. He began 130mg/kg/day of dC and dT in August of 2015 and increased to 260 mg/kg/dayin February of 2016. He has shown improved energy.

There were no observed adverse effects.

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1.-39. (canceled)
 40. A method of treating thymidine kinase 2 (TK2)deficiency in a human subject in need thereof comprising administeringto the subject a therapeutically effective amount of a compositioncomprising a mixture of deoxycytidine (dC) and deoxythymidine (dT),wherein the therapeutically effective amount is between about 50mg/kg/day and about 500 mg/kg/day of total deoxynucleoside in thecomposition and the composition is administered to the subjectparenterally.
 41. The method of claim 40, wherein the parenteraladministration is intravenous administration.
 42. The method of claim40, wherein the composition is administered one time a day, two times aday, three times daily, four times daily, five times daily or six timesdaily.
 43. A method of treating thymidine kinase 2 (TK2) deficiency in ahuman subject in need thereof comprising administering to the subject atherapeutically effective amount of a composition comprising a mixtureof deoxycytidine (dC) and deoxythymidine (dT), wherein thetherapeutically effective amount is between about 50 mg/kg/day and about120 mg/kg/day of total deoxynucleoside in the composition and thecomposition is administered to the subject parenterally.
 44. The methodof claim 43, wherein the parenteral administration is intravenousadministration.
 45. The method of claim 43, wherein the composition isadministered one time a day, two times a day, three times daily, fourtimes daily, five times daily or six times daily.
 46. The method ofclaim 45, wherein the therapeutically effective amount of thecomposition per administration is between about 8 mg/kg to about 20mg/kg.
 47. The method of claim 40, wherein the ratio of deoxycytidine(dC) and deoxythymidine (dT) is 50/50, 5/95, 10/90, 15/85, 20/80, 25/75,30/70, 35/65, 40/60, 45/55, 55/45, 60/40, 65/35, 70/30, 75/25, 80/20,85/15, 90/10, or 95/5.
 48. The method of claim 40, wherein the ratio ofdeoxycytidine (dC) and deoxythymidine (dT) is 50/50.
 49. The method ofclaim 43, wherein the ratio of deoxycytidine (dC) and deoxythymidine(dT) is 50/50, 5/95, 10/90, 15/85, 20/80, 25/75, 30/70, 35/65, 40/60,45/55, 55/45, 60/40, 65/35, 70/30, 75/25, 80/20, 85/15, 90/10, or 95/5.50. The method of claim 43, wherein the ratio of deoxycytidine (dC) anddeoxythymidine (dT) is 50/50.
 51. A method of treating mitochondrial DNAdepletion syndrome in a human subject in need thereof comprisingadministering to the subject a therapeutically effective amount of acomposition comprising at least one deoxynucleoside, wherein themitochondrial DNA depletion syndrome is characterized by at least onemutation in a gene chosen from the group consisting of: DGUOK; TYMP; andRRM2B and wherein the therapeutically effective amount is between about50 mg/kg/day and about 500 mg/kg/day of total deoxynucleoside in thecomposition and the composition is administered to the subjectparenterally.
 52. The method of claim 51, wherein the composition isadministered intravenously.
 53. The method of claim 51, wherein thecomposition is administered one time a day, two times a day, three timesdaily, four times daily, five times daily or six times daily.
 54. Amethod of treating mitochondrial DNA depletion syndrome in a humansubject in need thereof comprising administering to the subject atherapeutically effective amount of a composition comprising at leastone deoxynucleoside, wherein the mitochondrial DNA depletion syndrome ischaracterized by at least one mutation in a gene chosen from the groupconsisting of: DGUOK; TYMP; and RRM2B and wherein the therapeuticallyeffective amount is between about 50 mg/kg/day and about 120 mg/kg/dayof total deoxynucleoside in the composition and the composition isadministered to the subject parenterally.
 55. The method of claim 54,wherein the composition is administered intravenously.
 56. The method ofclaim 54, wherein the composition is administered one time a day, twotimes a day, three times daily, four times daily, five times daily orsix times daily.
 57. The method of claim 56, wherein the therapeuticallyeffective amount of the composition per administration is between about8 mg/kg to about 20 mg/kg.
 58. A method of treating mitochondrial DNAdepletion syndrome in a human subject in need thereof comprisingadministering to the subject a therapeutically effective amount of acomposition comprising at least one deoxynucleoside, wherein themitochondrial DNA depletion syndrome is characterized by at least onemutation in a gene chosen from the group consisting of: TK2, DGUOK;TYMP; and RRM2B and wherein the therapeutically effective amount isbetween about 50 mg/kg/day and about 500 mg/kg/day of totaldeoxynucleoside in the composition and the composition is administeredto the subject intrathecally.